1. Field of the Invention
The invention generally relates to targeted gene therapy using recombinant vectors and particularly adenovirus vectors. The invention specifically relates to replication-conditional vectors and methods for using them. Such vectors are able to selectively replicate in a target tissue to provide a therapeutic benefit from the presence of the vector per se or from heterologous gene products expressed from the vector and distributed throughout the tissue. In such vectors, a gene essential for replication is placed under the control of a heterologous tissue-specific transcriptional regulatory sequence. Thus, replication is conditioned on the presence of a factor(s) that induces transcription or the absence of a factor(s) that inhibits transcription of the gene by means of the transcriptional regulatory sequence. With this vector, therefore, a target tissue can be selectively treated. The invention also relates to methods of using the vectors to screen a tissue for the presence or absence of transcriptional regulatory functions that permit vector replication by means of the transcriptional regulatory sequence. The invention also relates to cells for producing recombinant replication-conditional vectors useful for targeted gene therapy.
2. Background Art
One of the major goals for therapeutic use of exogenous genes has been cell targeting with high specificity. General approaches have included systemic introduction of DNA, DNA-protein complexes, and liposomes. In situ administration of retroviruses has also been used for cells that are actively replicating.
However, because of the lack of, or significantly low, cell-specificity and inefficient gene transfer, the targeting of desired genes to specific cells in an organism has been a major obstacle for exogenous gene-based therapy. Thus, the use of such genes has been limited.
Tumor cells are among those cell types for which it would be especially desirable to provide a means for exogenous gene targeting. In an embodiment of the present invention, compositions and methods are provide to deliver exogenous genes to tumor cells safely and efficiently.
Adenoviruses are nonenveloped, regular icosohedrons. The protein coat (capsid) is composed of 252 capsomeres of which 240 are hexons and 12 are pentons. Most of the detailed structural studies of the adenovirus polypeptides have been done for adenovirus types 2 and 5. The viral DNA is 23.85xc3x97106 daltons for adenovirus 2 and varies slightly in size depending on serotype. The DNA has inverted terminal repeats and the length of these varies with the serotype.
The replicative cycle is divided into early (E) and late (L) phases. The late phase defines the onset of viral DNA replication. Adenovirus structural proteins are generally synthesized during the late phase. Following adenovirus infection, host DNA and protein synthesis is inhibited in cells infected with most serotypes. The adenovirus lytic cycle with adenovirus 2 and adenovirus 5 is very efficient and results in approximately 10,000 virions per infected cell along with the synthesis of excess viral protein and DNA that is not incorporated into the virion. Early adenovirus transcription is a complicated sequence of interrelated biochemical events, but it entails essentially the synthesis of viral RNAs prior to the onset of viral DNA replication.
The organization of the adenovirus genome is similar in all of the adenovirus groups and specific functions are generally positioned at identical locations for each serotype studied. Early cytoplasmic messenger RNAs are complementary to four defined, noncontiguous regions on the viral DNA. These regions are designated (E1-E4). The early transcripts have been classified into an array of immediate early (E1a), delayed early (E1b, E2a, E2b, E3 and E4), and intermediate (IVa2.IX) regions.
The E1a region is involved in transcriptional transactivation of viral and cellular genes as well as transcriptional repression of other sequences. The E1a gene exerts an important control function on all of the other early adenovirus messenger RNAs. In normal tissues, in order to transcribe regions E1b, E2a, E2b, E3, or E4 efficiently, active E1a product is required. However, as discussed below, the E1a function may be bypassed. Cells may be manipulated to provide E1a-like functions or may naturally contain such functions. The virus may also be manipulated to bypass the functions as described below.
The E1b region is required for the normal progression of viral events late in infection. The E1b product acts in the host nucleus. Mutants generated within the E1b sequences exhibit diminished late viral mRNA accumulation as well as impairment in the inhibition of host cellular transport normally observed late in adenovirus infection (Berkner, K. L., Biotechniques 6:616-629 (1988)). E1b is required for altering functions of the host cell such that processing and transport are shifted in favor of viral late gene products. These products then result in viral packaging and release of virions. E1b produces a 19 kD protein that prevents apoptosis. E1b also produces a 55 kD protein that binds to p53.
For a complete review on adenoviruses and their replication, see Horwitz, M. S., Virology 2d ed., Fields, B. N., eds., Raven Press Limited, New York (1990), Chapter 60, pp. 1679-1721.
Adenovirus as Recombinant Delivery Vehicle
Adenovirus provides advantages as a vector for adequate gene delivery for the following reasons. It is a double stranded DNA nonenveloped virus with tropism for the human respiratory system and gastrointestinal tract. It causes a mild flu-like disease. Adenoviral vectors enter cells by receptor mediated endocytosis. The large (36 kilobase) genome allows for the removal of genes essential for replication and nonessential regions so that foreign DNA may be inserted and expressed from the viral genome. Adenoviruses infect a wide variety of cell types in vivo and in vitro. Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes. The expression of viral or foreign genes from the adenovirus genome does not require a replicating cell. Adenovirus vectors rarely integrate into the host chromosome; the adenovirus genome remains as an extrachromosomal element in the cellular nucleus. There is no association of human malignancy with adenovirus infection; attenuated strains have been developed and have been used in humans for live vaccines.
For a more detailed discussion of the use of adenovirus vectors for gene therapy, see Berkner, K. L., Biotechniques 6:616-629 (1988); Trapnell, B. C., Advanced Drug Delivery Reviews 12:185-199 (1993).
Adenovirus vectors are generally deleted in the E1 region of the virus. The E1 region may then be substituted with the DNA sequences of interest. It was pointed out in a recent article on human gene therapy, however, that xe2x80x9cthe main disadvantage in the use of adenovirus as a gene transfer vector is that the viral vector generally remains episomal and does not replicate, thus, cell division leads to the eventual loss of the vector from the daughter cellsxe2x80x9d (Morgan, R. A., et al., Annual Review of Biochemistry 62:191-217 (1993)) (emphasis added).
Non-replication of the vector leads not only to eventual loss of the vector without expression in most or all of the target cells but also leads to insufficient expression in the cells that do take up the vector, because copies of the gene whose expression is desired are insufficient for maximum effect. The insufficiency of gene expression is a general limitation of all non-replicating delivery vectors. Thus, it is desirable to introduce a vector that can provide multiple copies of a gene and hence greater amounts of the product of that gene. The present invention overcomes the disadvantages discussed above by providing a tissue-specific, and especially a tumor-specific replicating vector, multiple DNA copies, and thus increased amounts of gene product.
Adenoviral vectors for recombinant gene expression have been produced in the human embryonic kidney cell line 293 (Graham, F. L. et al., J. Gen. Virol. 36:59-72 (1977)). This cell line, initially transformed with human adenovirus 5, now contains the left end of the adenovirus 5 genome and expresses E1. Therefore, these cells are permissive for growth of adenovirus 2 and adenovirus 5 mutants defective in E1 functions. They have been extensively used for the isolation and propagation of E1 mutants. Therefore, 293 cells have been used for helper-independent cloning and expression of adenovirus vectors in mammalian cells. E1 genes integrated in cellular DNA of 293 cells are expressed at levels which permit deletion of these genes from the viral vector genome. The deletion provides a nonessential region into which DNA may be inserted. For a review, see, Young, C. S. H., et al. in The Adenoviruses, Ginsberg, H. S., ed., Plenum Press, New York and London (1984), pp. 125-172.
However, 293 cells are subject to severe limitations as producer cells for adenovirus vectors. Growth rates are low. Titres are limited, especially when the vector produces a heterologous gene product that proves toxic for the cells. Recombination with the viral E1 sequence in the genome can lead to the contamination of the recombinant defective virus with unsafe wild-type virus. The quality of certain viral preparations is poor with regard to the ratio of virus particle to plaque forming unit. Further, the cell line does not support growth of more highly deleted mutants because the expression of E1 in combination with other viral genes in the cellular genome (required to complement the further deletion), such as E4, is toxic to the cells. Therefore, the amount of heterologous DNA that can be inserted into the viral genome is limited in these cells. It is desirable, therefore, to produce adenovirus vectors for gene therapy in a cell that cannot produce wild-type recombinants and can produce high titres of high-equality virus. The present invention overcomes these limitations.
In view of the limitations discussed above, a general object of the invention is to provide novel vectors for tissue-specific vector replication and gene expression from the replicating vector. Accordingly, the invention is directed to a vector that contains a gene which is essential for replication, and which gene is operably linked to a heterologous transcriptional regulatory sequence, such that a vector is created whose replication is conditioned upon the presence of a trans-acting transcriptional regulatory factor(s) that permits transcription from the transcriptional regulatory sequence, or the absence of a transcriptional regulatory factor(s) that normally prevents transcription from that transcriptional regulatory sequence. Thus, these regulatory sequences are specifically activated or derepressed in the target tissue so that replication of the vector proceeds in that tissue.
Another object of the invention is to provide tissue-specific treatment of an abnormal tissue. Thus, a further object of the invention is to provide a method to selectively distribute a vector in vivo in a target tissue, such that a greater number of cells are treated than would be treated with a non-replicating vector, and treatment is avoided or significantly reduced in non-target tissue. Accordingly, a method is provided for selectively distributing a vector in a target tissue by introducing the replication-conditional vector of the present invention into a target tissue that contains a transcriptional regulatory factor(s) that allows replication of the vector or is deficient in a transcription-inhibiting factor(s) that prevents replication of the vector.
For providing tissue-specific treatment, another object of the invention is to selectively distribute a polynucleotide in a target tissue in vivo. Accordingly, the invention is directed to a method for selectively distributing a polynucleotide in a target tissue in vivo by introducing the replication-conditional vector of the present invention, containing the polynucleotide, into the target tissue that contains a transcriptional regulatory factor(s) that allows replication of the vector or is deficient in a transcription-inhibiting factor(s) that prevents replication of the vector.
For providing tissue-specific treatment, a another object of the invention is to selectively distribute a heterologous gene product in a target tissue. Accordingly, the replication-conditional vectors of the present invention are constructed so that they contain a heterologous DNA sequence encoding a gene product that is expressed in the vector. When the vector replicates in the target tissue, effective quantities of the desired gene product are also produced in the target tissue.
Another object of the invention is to provide a method to identify abnormal tissue that can be treated by the vectors of the present invention. Therefore, a further object of the invention is to identify a tissue in which the replication-conditional vectors of the present invention can be replicated by means of the transcriptional regulatory sequence contained on the vector. Accordingly, the invention is further directed to a method wherein the replication-conditional vectors of the present invention are exposed to a given abnormal tissue. If that tissue contains a transcriptional regulatory factor(s) that allows replication of the vector or is deficient in a transcription-inhibiting factor(s) that prevents replication of the vector, then replication of the vector will occur and can be detected. Following identification of such a tissue, targeted treatment of that tissue can be effected by tissue-specific transcription and the consequent vector replication in that tissue in vivo.
Thus, a method is provided for assaying vector utility for tissue treatment comprising the steps of removing a tissue biopsy from a patient, explanting the biopsy into tissue culture, introducing a replication-conditional vector into the cells of the biopsy, and assaying for vector replication in the cells.
Another object of the invention is to provide producer cell lines for vector production. Preferably, the cell lines have one or more of the following characteristics: high titer virus production, resistance to toxic effects due to heterologous gene products expressed in the vector, lack of production of wild-type virus contaminating the virus preparation and resulting from recombination between integrated viral sequences and vector sequences, growth to high density and in suspension, unlimited passage potential, high growth rate, and by permitting the growth of highly deleted viruses that are impaired for viral functions and able to accommodate large pieces of heterologous DNA.
Accordingly, in a further embodiment of the invention, a cell line is provided containing the replication-conditional vector of the present invention, the cells of which cell line contain a transcriptional regulatory factor(s) that allows replication of the vector or is deficient in a transcription-inhibiting factor(s) that prevents replication of the vector.
In further embodiments of the invention, the cell line contains nucleic acid copies of the replicated vector. In other embodiments, the cell line contains virions produced in the cell by replication in the cell of the replication-conditional vector.
In further embodiments, a method is provided for producing a replication-conditional vector or virion comprising the steps of culturing the producer cell line described above and recovering the vector or virion from the cells. In still further embodiments, a method is provided for producing replication-conditional virions free of wild-type virions or viral vectors free of wild-type vectors, comprising the steps of culturing the producer cell line described above and recovering the replication-deficient virions or vectors from the cells.
In a preferred methods of treatment and diagnosis, the tissue is abnormally proliferating, and especially is tumor tissue. However, the methods are also directed to other abnormal tissue as described herein.
In preferred embodiments of the invention, the replication-conditional vector is a DNA tumor viral vector. In a further preferred embodiment, the DNA tumor viral vector is a vector selected from the group consisting of herpesvirus, papovavirus, papillomavirus, parvovirus, and hepatitis virus vectors. In a most preferred embodiment, the vector is an adenovirus vector. However, it is to be understood that potentially any vector source is useful if it contains a gene essential for replication that can be operably linked to a tissue-specific transcriptional regulatory sequence.
In further methods of treatment and diagnosis, the vector is introduced into the tissue by infection.
Replication can be vector nucleic acid replication alone or can also include virus replication (i.e., virion production). Thus, either DNA or virions or both may be distributed in the target tissue.
In a further preferred embodiment of the invention, a gene in the adenovirus E1 region is operably linked to the tissue-specific transcriptional regulatory sequence. Preferably, the E1a or E1b gene is operably linked to the tissue-specific transcriptional regulatory sequence.
In a further embodiment of the invention, the vector encodes a heterologous gene product. This heterologous gene product is expressed from the vector replicating in the target tissue.
In a further embodiment of the methods of treatment, the heterologous gene product is toxic for the target tissue.
In a further embodiment of the methods, the heterologous gene product acts on a non-toxic prodrug, converting the non-toxic prodrug into a form that is toxic for the target tissue. Preferably, the toxin has anti-tumor activity or eliminates cell proliferation.
In preferred embodiments of the invention, the transcriptional regulatory sequence is a promoter. Preferred promoters include, but are not limited to, carcinoembryonic antigen (CEA), DE3, xcex1-fetoprotein (AFP), Erb-B2, surfactant, and especially lung surfactant, and the tyrosinase promoter. However, any genetic control region that controls transcription of the essential gene can be used to activate (or derepress) the gene. Thus, other genetic control elements, such as enhancers, repressible sequences, and silencers, can be used to regulate replication of the vector in the target cell. The only requirement is that the genetic element be activated, derepressed, enhanced, or otherwise genetically regulated by factors in the host cell and, with respect to methods of treatment, not in the non-target cell.
Preferred enhancers include the DF3 breast cancer-specific enhancer and enhancers from viruses and the steroid receptor family. Other preferred transcriptional regulatory sequences include NF1, SP1, AP1, and FOS/JUN.
In further embodiments, promoters are not necessarily activated by factors in the target tissue, but are derepressed by factors present in the target tissue. Thus, in the target tissue, repression is lifted.
Transcriptional regulatory factors include, but are not limited to, transactivating factors produced by endogenous viral sequences such as from cytomegalovirus (CMV), HIV, Epstein-Barr virus (EBV), Herpes simplex virus (HSV), SV40, and other such viruses that are pathogenic in mammals and, particularly, in humans.
Methods for making such vectors are well known to the person of ordinary skill in the art. The art adequately teaches the construction of recombinant vectors with deletions or modifications in specific coding sequences and operable linkage to a heterologous transcription control sequence such that expression of a desired coding region is under control of the heterologous transcriptional regulatory sequence. Many viral sequences have been adequately mapped such that it is routine to identify a gene of choice and use appropriate well known techniques (such as homologous recombination of the virus with deleted or otherwise modified plasmids) to construct the vectors for tissue-specific replication and expression.